Gastrointestinal vascular permeability changes following spinal cord injury.

BACKGROUND: Gastrointestinal (GI) dysfunction is observed clinically after spinal cord injury (SCI) and contributes to the diminished long-term quality of life. Our study examined the acute and chronic GI vascular changes that occur following SCI. We demonstrated that the GI vascular tract in SCI mice becomes compromised during the acute phase of injury and persists into the chronic phase of injury. METHODS: Gastrointestinal vasculature permeability was measured using dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) at 48 hours, and 2 and 4 weeks following contusion spinal cord injury. Angiopoietin-1, a vascular stabilizing protein, was administered intravenously following injury. Intestinal contractile activity assessments were performed following the last imaging session. KEY RESULTS: Our results indicated that a single administration of Ang-1 reduced vascular permeability at 48 hours but the effect was only transient. However, when the treatment paradigm was changed from a single administration to multiple administrations of Ang-1 following contusion injury, our DCE MRI data indicated a significant decrease in GI vascular permeability 4 weeks after injury compared with vehicle control treated animals. This improved GI vascular permeability was associated with improved sustained intestinal contractile activity. We also demonstrated that Ang-1 reduced the expression of sICAM-1 in the ileum compared with the saline-treated group. CONCLUSIONS AND INFERENCES: We show that the GI vasculature is compromised in the acute and chronic phase of injury following spinal contusion. Our results also indicate that multiple administrations of Ang-1 can attenuate GI vascular permeability, possibly reduce inflammation, and improve sustained agonist-induced contraction compared with saline treatment.

Enhancing Therapeutic Efficacy of Oncolytic Herpes Simplex Virus-1 with Integrin ß1 Blocking Antibody OS2966.

Integrin ß1 receptor, expressed on the surface of tumor cells and macrophages in the tumor microenvironment (TME), has been implicated in both tumor progression and resistance to multiple modalities of therapy. OS2966 is the first clinical-ready humanized monoclonal antibody to block integrin ß1 and was recently orphan designated by the FDA Office of Orphan Products Development. Here, we tested therapeutic potential of OS2966-mediated integrin ß1 blockade to enhance the efficacy of oncolytic herpes simplex virus-1 (oHSV) through evaluation of virus replication, tumor cell killing efficiency, effect on the antiviral signaling pathway, co-culture assays of oHSV-infected cells with macrophages, and in vivo bioluminescence imaging on mammary fat pad triple-negative breast cancer xenograft and subcutaneous and intracranial glioma xenografts. OS2966 treatment decreased interferon signaling and proinflammatory cytokine induction in oHSV-treated tumor cells and inhibited migration of macrophages, resulting in enhanced oHSV replication and cytotoxicity. OS2966 treatment also significantly enhanced oHSV replication and oHSV-mediated antitumor efficacy in orthotopic xenograft models, including triple-negative breast cancer and glioblastoma. The results demonstrated the synergistic potential of the combinatory treatment approach with OS2966 to improve antitumor efficacy of conventional oHSV therapy.

Suppression of HMGB1 Released in the Glioblastoma Tumor Microenvironment Reduces Tumoral Edema.

HMGB1 is a ubiquitously expressed intracellular protein that binds DNA and transcription factors and regulates chromosomal structure and function. Under conditions of cell death or stress, it is actively or passively released by cells into the extracellular environment, where it functions as damage-associated molecular pattern (DAMP) that orchestrates pro-inflammatory cytokine release and inflammation. Our results demonstrate that HMGB1 is secreted in the tumor microenvironment after oncolytic HSV (oHSV) infection in vitro and in vivo. The impact of secreted HMGB1 on tumor growth and response to oncolytic viral therapy was evaluated by using HMGB1-blocking antibodies in vitro and in mice bearing intracranial tumors. IVIS and MRI imaging was utilized to visualize in real time virus spread, tumor growth, and changes in edema in mice. Our data showed that HMGB1 released in tumor microenvironment orchestrated increased vascular leakiness and edema. Further HMGB1 blocking antibodies rescued vascular leakiness and enhanced survival of intracranial glioma-bearing mice treated with oHSV.

Acute White Matter Tract Damage after Frontal Mild Traumatic Brain Injury.

Our understanding of mild traumatic brain injury (mTBI) is still in its infancy and to gain a greater understanding, relevant animal models should replicate many of the features seen in human mTBI. These include changes to diffusion tensor imaging (DTI) parameters, absence of anatomical lesions on conventional neuroimaging, and neurobehavioral deficits. The Maryland closed head TBI model causes anterior-posterior plus sagittal rotational acceleration of the brain, frequently observed with motor vehicle and sports-related TBI injuries. The injury reflects a concussive injury model without skull fracture. The goal of our study was to characterize the acute (72 h) pathophysiological changes occurring following a single mTBI using magnetic resonance imaging (MRI), behavioral assays, and histology. We assessed changes in fractional anisotropy (FA), mean (MD), longitudinal (LD), and radial (RD) diffusivities relative to pre-injury baseline measures. Significant differences were observed in both the longitudinal and radial diffusivities in the fimbria compared with baseline. A significant difference in radial diffusivity was also observed in the splenium of the corpus callosum compared with baseline. The exploratory activity of the mTBI animals was also assessed using computerized activity monitoring. A significant decrease was observed in ambulatory distance, average velocity, stereotypic counts, and vertical counts compared with baseline. Histological examination of the mTBI brain sections indicated a significant decrease in the expression of myelin basic protein in the fimbria, splenium, and internal capsule. Our findings demonstrate the vulnerability of the white matter tracts, specifically the fimbria and splenium, and the ability of DTI to identify changes to the integrity of the white matter tracts following mTBI.

Chronic cocaine administration causes extensive white matter damage in brain: diffusion tensor imaging and immunohistochemistry studies.

The effect of chronic cocaine exposure on multiple white matter structures in rodent brain was examined using diffusion tensor imaging (DTI), locomotor behavior, and end point histology. The animals received either cocaine at a dose of 100mg/kg (N=19), or saline (N=17) for 28 days through an implanted osmotic minipump. The animals underwent serial DTI scans, locomotor assessment, and end point histology for determining the expressions of myelin basic protein (MBP), neurofilament-heavy protein (NF-H), proteolipid protein (PLP), Nogo-A, aquaporin-4 (AQP-4), and growth associated protein-43 (GAP-43). Differences in the DTI measures were observed in the splenium (scc) and genu (gcc) of the corpus callosum (cc), fimbria (fi), and the internal capsule (ic). A significant increase in the activity in the fine motor movements and a significant decrease in the number of rearing events were observed in the cocaine-treated animals. Reduced MBP and Nogo-A and increased GAP-43 expressions were most consistently observed in these structures. A decrease in the NF-H expression was observed in fi and ic. The reduced expression of Nogo-A and the increased expression of GAP-43 may suggest destabilization of axonal connectivity and increased neurite growth with aberrant connections. Increased GAP-43 suggests drug-induced plasticity or a possible repair mechanism response. The findings indicated that multiple white matter tracts are affected following chronic cocaine exposure.

Optic nerve sheath diameter measurement techniques: examination using a novel ex-vivo porcine model.

BACKGROUND: Ultrasound (U/S) and MRI measurements of the optic nerve sheath diameter (ONSD) have been proposed as intracranial pressure measurement surrogates, but these methods have not been fully evaluated or standardized. The purpose of this study was to develop an ex-vivo model for evaluating ONSD measurement techniques by comparing U/S and MRI measurements to physical measurements. METHODS: The left eye of post mortem juvenile pigs (N = 3) was excised and the subdural space of the optic nerve cannulated. Caliper measurements and U/S imaging measurements of the ONSD were acquired at baseline and following 1 cc saline infusion into the sheath. The samples were then embedded in 0.5% agarose and imaged in a 7 Tesla (7T) MRI. The ONSD was subsequently measured with digital calipers at locations and directions matching the U/S and direct measurements. RESULTS: Both MRI and sonographic measurements were in agreement with direct measurements. U/S data, especially axial images, exhibited a positive bias and more variance (bias: 1.318, 95% limit of agreement: 8.609) compared to MRI (bias: 0.3156, 95% limit of agreement: 2.773). In addition, U/S images were much more dependent on probe placement, distance between probe and target, and imaging plane. CONCLUSIONS: This model appears to be a valid test-bed for continued scrutiny of ONSD measurement techniques. In this model, 7T MRI was accurate and potentially useful for in-vivo measurements where direct measurements are not available. Current limitations with ultrasound imaging for ONSD measurement associated with image acquisition technique and equipment necessitate further standardization to improve its clinical utility.

Behavioral and histopathological alterations resulting from mild fluid percussion injury.

The majority of people who sustain a traumatic brain injury (TBI) have an injury that can be classified as mild (often referred to as concussion). Although head CT scans for most subjects who have sustained a mild TBI (mTBI) are negative, these persons may still suffer from neurocognitive and neurobehavioral deficits. In order to expedite pre-clinical research and develop therapies, there is a need for well-characterized animal models of mTBI that reflect the neurological, neurocognitive, and pathological changes seen in human patients. In the present study, we examined the motor, cognitive, and histopathological changes resulting from 1.0 and 1.5 atmosphere (atm) overpressure fluid percussion injury (FPI). Both 1.0 and 1.5 atm FPI injury caused transient suppression of acute neurological functions, but did not result in visible brain contusion. Animals injured with 1.0 atm FPI did not show significant motor, vestibulomotor, or learning and memory deficits. In contrast, 1.5 atm injury caused transient motor disturbances, and resulted in a significant impairment of spatial learning and short-term memory. In addition, 1.5 atm FPI caused a marked reduction in cerebral perfusion at the site of injury that lasted for several hours. Consistent with previous studies, 1.5 atm FPI did not cause visible neuronal loss in the hippocampus or in the neocortex. However, a robust inflammatory response (as indicated by enhanced GFAP and Iba1 immunoreactivity) in the corpus callosum and the thalamus was observed. Examination of fractional anisotropy color maps after diffusion tensor imaging (DTI) revealed a significant decrease of FA values in the cingulum, an area found to have increased silver impregnation, suggesting axonal injury. Increased silver impregnation was also observed in the corpus callosum, and internal and external capsules. These findings are consistent with the deficits and pathologies associated with mild TBI in humans, and support the use of mild FPI as a model to evaluate putative therapeutic options.

Accuracy and resolution of in vitro imaging based porcine lens volumetric measurements.

There is considerable interest in determining lens volume in the living eye. Lens volume is of interest to understand accommodative changes in the lens and to size accommodative IOLs (A-IOLs) to fit the capsular bag. Some studies have suggested lens volume change during accommodation. Magnetic Resonance Imaging (MRI) is the only method available to determine lens volume in vivo. MRI is, by its nature, relatively low in temporal and spatial resolution. Therefore analysis often requires determining lens volume from single image slices with relatively low resolution on which only simple image analysis methods can be used and without repeated measures. In this study, 7 T MRI scans encompassing the full lens volume were performed on 19 enucleated pig eyes. The eyes were then dissected to isolate and photograph the lens in profile and the lens volumes were measured empirically using a fluid displacement method. Lens volumes were calculated from two- and three-dimensional (2D and 3D) MR and 2D photographic profile images of the isolated lenses using several different analysis methods. Image based and actual measured lens volumes were compared. The average image-based volume of all lenses varied from the average measured volume of all lenses by 0.6%-6.4% depending on the image analysis method. Image analysis methods that use gradient based edge detection showed higher precision with actual volumes (r(2): 0.957-0.990), while threshold based segmentation had poorer correlations (r(2): 0.759-0.828). The root-mean-square (RMS) difference between image analysis based volumes and fluid displacement measured volumes ranged from 8.51 µl to 25.79 µl. This provides an estimate of the error of previously published methods used to calculate lens volume. Immobilized, enucleated porcine eyes permit improved MR image resolution relative to living eyes and therefore improved image analysis methods to calculate lens volume. The results show that some of the accommodative changes in lens volume reported in the literature are likely below the resolution limits of imaging methods used. MRI, even with detailed image analysis methods used here, is unlikely to achieve the resolution required to accurately size an A-IOL to the capsular bag.

Effects of optical defocus on refractive development in monkeys: evidence for local, regionally selective mechanisms.

PURPOSE: To characterize the influence of optical defocus on ocular shape and the pattern of peripheral refraction in infant rhesus monkeys. METHODS: Starting at 3 weeks of age, eight infant monkeys were reared wearing -3 diopter (D) spectacle lenses over one eye that produced relative hyperopic defocus in the nasal field (NF) but allowed unrestricted vision in the temporal field (NF group). Six infants were reared with monocular -3 D lenses that produced relative hyperopic defocus across the entire field of view. Control data were obtained from 11 normal monkeys. Refractive development was assessed by streak retinoscopy performed along the pupillary axis and at eccentricities of 15 degrees, 30 degrees, and 45 degrees along the vertical and horizontal meridians. Central axial dimensions and eye shape were assessed with magnetic resonance imaging. RESULTS: In response to full-field hyperopic defocus, the eye developed relative central axial myopia, became less oblate, and exhibited relative peripheral hyperopia in both the nasal and the temporal hemifields. Conversely, nasal-field hyperopic defocus produced relative myopia that was largely restricted to the nasal hemifield; these alterations in the patterns of peripheral refraction in the NF monkeys were associated with local, region-specific alterations in vitreous chamber depth in the treated hemiretina. CONCLUSIONS: Optically imposed defocus can alter the shape and pattern of peripheral refraction in infant primates. Like those of form deprivation, the effects of optical defocus in primates are dominated by mechanisms that integrate visual signals in a spatially restricted manner and exert their influence in a regionally selective manner.

Hemiretinal form deprivation: evidence for local control of eye growth and refractive development in infant monkeys.

PURPOSE: To determine whether refractive development in primates is mediated by local retinal mechanisms, the authors examined the effects of hemiretinal form deprivation on ocular growth and the pattern of peripheral refractions in rhesus monkeys. METHODS: Beginning at approximately 3 weeks of age, nine infant monkeys were reared wearing monocular diffuser lenses that eliminated form vision in the nasal field (nasal field diffuser [NFD]). Control data were obtained from the nontreated fellow eyes, 24 normal monkeys, and 19 monkeys treated with full-field diffusers. Refractive development was assessed by retinoscopy performed along the pupillary axis and at eccentricities of 15 degrees, 30 degrees, and 45 degrees. Central axial dimensions and eye shape were assessed by A-scan ultrasonography and magnetic resonance imaging, respectively. RESULTS: Hemiretinal form deprivation altered refractive development in a regionally selective manner, typically producing myopia in the treated hemifields. In particular, six of the NFD monkeys exhibited substantial amounts (-1.81 to -9.00 D) of relative myopia in the nasal field that were most obvious at the 15 degrees and 30 degrees nasal field eccentricities. The other three NFD monkeys exhibited small amounts of relative hyperopia in the treated field. The alterations in peripheral refraction were associated with local, region-specific alterations in vitreous chamber depth in the treated hemiretina. CONCLUSIONS: The effects of form deprivation on refractive development and eye growth in primates are mediated by mechanisms, presumably retinal, that integrate visual signals in a spatially restricted manner and exert their influence locally.

Effects of form deprivation on peripheral refractions and ocular shape in infant rhesus monkeys (Macaca mulatta).

PURPOSE: To determine whether visual experience can alter ocular shape and peripheral refractive error pattern, the authors investigated the effects of form deprivation on refractive development in infant rhesus monkeys. METHODS: Monocular form deprivation was imposed in 10 rhesus monkeys by securing diffuser lenses in front of their treated eyes between 22 +/- 2 and 163 +/- 17 days of age. Each eye's refractive status was measured longitudinally by retinoscopy along the pupillary axis and at 15 degrees intervals along the horizontal meridian to eccentricities of 45 degrees . Control data for peripheral refraction were obtained from the nontreated fellow eyes and six untreated monkeys. Near the end of the diffuser-rearing period, the shape of the posterior globe was assessed by magnetic resonance imaging. Central axial dimensions were also determined by A-scan ultrasonography. RESULTS: Form deprivation produced interocular differences in central refractive errors that varied between +2.69 and -10.31 D (treated eye-fellow eye). All seven diffuser-reared monkeys that developed at least 2.00 D of relative central axial myopia also showed relative hyperopia in the periphery that increased in magnitude with eccentricity. Alterations in peripheral refraction were highly correlated with eccentricity-dependent changes in vitreous chamber depth and the shape of the posterior globe. CONCLUSIONS: Like humans with myopia, monkeys with form-deprivation myopia exhibit relative peripheral hyperopia and eyes that are less oblate and more prolate. Thus, in addition to producing central refractive errors, abnormal visual experience can alter the shape of the posterior globe and the pattern of peripheral refractive errors in infant primates.

Bax shuttling after neonatal hypoxia-ischemia: hyperoxia effects.

Perinatal hypoxia-ischemia (HI) occurs in 0.2%-0.4% of all live births, with 100% O(2) resuscitation (HHI) remaining a standard clinical treatment. HI produces a broad spectrum of neuronal death phenotypes ranging from a more noninflammatory apoptotic death to a more inflammatory necrotic cell death that may be responsible for the broad spectrum of reported dysfunctional outcomes. However, the mechanisms that would account for this phenotypic spectrum of cell death are not fully understood. Here, we provide evidence that Bcl-2-associated X protein (Bax) can shuttle to different subcellular compartments in response to HI, thus triggering the different organelle-associated cell death signaling cascades resulting in cell death phenotype diversity. There was an early increase in intranuclear and total nuclear Bax protein levels followed by a later Bax redistribution to the mitochondria and endoplasmic reticulum (ER). Associated with the organelle-specific Bax shuttling time course, there was an increase in nuclear phosphorylated p53, cytosolic cleaved caspase-3, and caspase-12. When HI-treated P7 rats were resuscitated with 100% O(2) (HHI), there were increased lesion volumes as determined by T2-weighted magnetic resonance imaging with no change in cortical apoptotic signaling compared with HI treatment alone. There was, however, increased inflammatory (cytosolic-cleaved interleukin-1beta) and necrotic (increased nuclear 55-kDa-cleaved PARP-1 [poly-ADP-ribose 1] and decreased nuclear HMGB1 [nuclear high-mobility group box 1]) after HHI. Furthermore, HHI increased ER calpain activation and ER Bax protein levels compared with HI alone. These data suggest that 100% O(2) resuscitation increases Bax-mediated activation of ER cell death signaling, inflammation, and lesion volume by increasing necrotic-like cell death. In light of these findings, the use of 100% O(2) treatment for neonatal HI should be reevaluated.

Cortical reorganization in NT3-treated experimental spinal cord injury: Functional magnetic resonance imaging.

Functional magnetic resonance imaging (fMRI) studies were performed for visualizing ongoing brain plasticity in Neurotrophin-3 (NT3)-treated experimental spinal cord injury (SCI). In response to the electrical stimulation of the forepaw, the NT3-treated animals showed extensive activation of brain structures that included contralateral cortex, thalamus, caudate putamen, hippocampus, and periaqueductal gray. Quantitative analysis of the fMRI data indicated significant changes both in the volume and center of activations in NT3-treated animals relative to saline-treated controls. A strong activation in both ipsi- and contralateral periaqueductal gray and thalamus was observed in NT3-treated animals. These studies indicate ongoing brain reorganization in the SCI animals. The fMRI results also suggest that NT3 may influence nociceptive pathways.

Functional magnetic resonance imaging in rodents: Methodology and application to spinal cord injury.

Functional MRI (fMRI) on spinal cord-injured rodents at 4 and 8 weeks post injury (PI) is described. The paradigm for fMRI, based on electrical stimulation of rat paws, was automated using an in-house designed microprocessor-based controller that was interfaced to a stimulator. The MR images were spatially normalized to the Paxinos and Watson atlas using publicly available digital images of the cryosections. In normal uninjured animals, the activation was confined to the contralateral somatosensory cortex. In contrast, in injured animals, extensive activation, which included structures such as ipsilateral cortex, thalamus, hippocampus, and the caudate putamen, was observed at 4 and 8 weeks PI. Quantitative cluster analysis was carried out to calculate the volumes and centers of activation in individual brain structures. Based on this analysis, significant increase in activation between 4 and 8 weeks was observed only in the ipsilateral caudate putamen and thalamus. These studies suggest extensive and ongoing brain reorganization in spinal cord-injured animals.

Cortical spreading depression in the feline brain following sustained and transient stimuli studied using diffusion-weighted imaging.

Cortical spreading depression (CSD) was induced by transient (10 min) applications of KCl in agar upon the cortical surface of alpha-chloralose anaesthetised cats. Its features were compared with CSD resulting from sustained applications of crystalline KCl through a mapping of the apparent diffusion coefficient (ADC) using diffusion-weighted echo planar imaging (DWI) over a poststimulus period of 60-100 min. Individual CSD events were computationally detected with the aid of Savitzky-Golay smoothing applied to critically sampled data derived from regions of interest (ROIs) made up of 2 x 2 pixel matrices. The latter were consistently placed at three selected sites on the suprasylvian gyrus (SG) and six sites on the marginal gyrus (MG). The CSD events thus detected were then quantitatively characterised for each ROI using the original time series. Both stimuli consistently elicited similar spreading patterns of initial, primary CSD events that propagated over the SG and marginal MG and were restricted to the hemispheres on which the stimuli were applied. There followed secondary events over smaller extents of cortical surface. Sustained stimuli elicited primary and secondary CSD events with similar amplitudes of ADC deflection that were distributed around a single mean. The ADC deflections were also conserved in peak amplitude throughout the course of their propagation. The initial primary event showed a poststimulus latency of 1.1 +/- 0.1 min. Successive secondary events followed at longer, but uniform, time intervals of around 10 min. Primary and secondary CSDs showed significantly different velocities of conduction (3.32 +/- 0.43 mm min(-1) vs. 2.11 +/- 0.21 mm min(-1), respectively; n = 5) across the cerebral hemisphere. In contrast, transient stimuli produced significantly fewer numbers of CSD events (3.8 +/- 0.5 events per animal, n = 5) than did sustained stimuli (7.4 +/- 0.5 events per animal, mean +/- S.E.M., n = 5, P = 0.002). The peak ADC deflection of their primary CSD events declined by approximately 30 % as they propagated from their initiation site to the interhemispheric boundary. The primary CSD event following a transient stimulus showed a latency of 1.4 +/- 0.1 min. It was followed by successive and smaller secondary ADC deflections that were separated by progressively longer time intervals. Conduction velocities of secondary events were similar to those of primary events. Conduction velocities of both primary and secondary events were slower than their counterparts following a sustained stimulus. ADC changes associated with CSD thus persist at times well after stimulus withdrawal and vary markedly with the nature of the initiating stimulus even in brain regions remote from the stimulus site.